Journal: medRxiv

Article Title: Cholangiocyte glycocalyx degradation boosts primary sclerosing cholangitis

doi: 10.1101/2024.06.27.24309484

Figure Lengend Snippet: Desialylation enhances bile acid induced toxicity in 2D and 3D cholangiocyte cultures. A Representative images of immunofluorescence stainings of cholangiocellular organoids and MMNK-1 cells. Stainings with DAPI (blue) and biotinylated lectins SNA (staining α2,6 linked sialic acid), MALII (staining α2,3 linked sialic acid) and PNA (staining β1,3 linked galactose) (red). Scale bars, 50 µM, nt = non treated B Viability of cholangiocellular organoids after treatment with Enterococcus faecalis (E.f.) sialidase, bile acid (GCDC) or treatment with GCDC after desialylation with E.f. sialidase. Assessed by the MTT assay. Experiments (n = 6) were performed in triplicate and data are shown as boxplots with mean ± standard error of the mean. **** p < 0.0001 one-way ANOVA, and post-hoc Dunnett’s multiple comparison tests. C Annexin V staining of cholangiocellular organoids analyzed via flow cytometry. Cells were treated with either E.f. sialidase, GCDC (bile acid) or a combination of both. Experiments (n = 4) were performed in triplicate and data are shown as boxplots with mean ± standard error of the mean. D Flow cytometrie histograms of the lectin staining of MMNK-1 cholangiocytes: live cells stained with lectins MALII (staining α2,3 linked sialic acid) and PNA (staining β1,3 linked galactose) with or without treatment with Streptococcus mitis sialidase A or B.

Article Snippet: Cells were incubated with the biotinylated lectins Maackia amurensis lectin (MAL-II, 0.005 mg/mL f.c., Vector Laboratories; detecting α2,3-linked sialic acid), Sambucus nigra agglutinin (SNA, 0.01 mg/mL f.c., Vector Laboratories; detecting α2,6-linked sialic acid) or Peanut agglutinin (PNA, 0.025 mg/ml f.c., Vector Laboratories; detecting β1,3-linked galactose) for one hour at room temperature in Tris buffered saline pH 7.5 with 1 mM MgCl2, 1 mM MnCl 2 and 1 mM CaCl 2 .

Techniques: Immunofluorescence, Staining, MTT Assay, Comparison, Flow Cytometry

Journal: Journal of Virology

Article Title: NKp46 O-Glycan Sequences That Are Involved in the Interaction with Hemagglutinin Type 1 of Influenza Virus ▿

doi: 10.1128/JVI.01815-09

Figure Lengend Snippet: O-Glycan analysis of COS-produced wild-type and mutant NKp46-Igs did not reveal any unique O-glycoform at position Thr225. NP-HPLC profiles of undigested glycans released from COS-produced NKp46-Ig (A), COS-produced NKp46-T225A-Ig (E) and NKp46-T125A-Ig (F) mutants are shown. Aliquots of the total 2AB-labeled glycan pool were further incubated with different exoglycosidases. Following digestion, the products were analyzed by NP-HPLC. Chromatograms shown in panels B to D represent NKp46-Ig glycans after digestion with sialidase A (B), sialidase S (C), and sialidase A plus galactosidase (D). The structures were allocated by their elution position measured in GU before and after digestion with exoglycosidases and reference to known GU values. The major peaks have been numbered and are described in detail in Table ​Table11.

Article Snippet: Exoglycosidases were used at the following concentrations: Arthrobacter ureafaciens sialidase (sialidase A, cleaves both α2,3 and α2,6 sialic acid residues) (ABS, EC 3.2.1.18), 1 to 2 U/ml; bovine testis β-galactosidase (BTG, EC3.2.1.23), 2U/ml; and Streptococcus pneumoniae sialidase (sialidase S, cleaves only α2,3 sialic acid residues) (EC 3.2.1.18, Nan1), 1 U/ml (all enzymes were from Glyco/Prozyme).

Techniques: Produced, Mutagenesis, Normal Phase Liquid Chromatography, Labeling, Incubation

Journal: Journal of Virology

Article Title: NKp46 O-Glycan Sequences That Are Involved in the Interaction with Hemagglutinin Type 1 of Influenza Virus ▿

doi: 10.1128/JVI.01815-09

Figure Lengend Snippet: O-Glycoforms of HEK293T- and CHO-produced NKp46. NP-HPLC profiles of undigested glycans released from HEK293T-NKp46-Ig (A) and CHO-NKp46-Ig (E) are shown. Aliquots of the total 2AB-labeled glycan pool were further incubated with different exoglycosidases. Following digestion, the products were analyzed by NP-HPLC. Chromatograms shown in panels B to D (HEK293T produced) and F to H (CHO produced) represent glycans after digestion with sialidase S (B and F), sialidase A (C and G), and sialidase A plus galactosidase (D and H). The structures were allocated by their elution positions measured in GU before and after digestion with exoglycosidases and with reference to known GU values. The major peaks have been numbered and are described in detail in Table ​Table11.

Article Snippet: Exoglycosidases were used at the following concentrations: Arthrobacter ureafaciens sialidase (sialidase A, cleaves both α2,3 and α2,6 sialic acid residues) (ABS, EC 3.2.1.18), 1 to 2 U/ml; bovine testis β-galactosidase (BTG, EC3.2.1.23), 2U/ml; and Streptococcus pneumoniae sialidase (sialidase S, cleaves only α2,3 sialic acid residues) (EC 3.2.1.18, Nan1), 1 U/ml (all enzymes were from Glyco/Prozyme).

Techniques: Produced, Normal Phase Liquid Chromatography, Labeling, Incubation